Impact of Normothermic Perfusion and Protein Supplementation on Human Endothelial Cell Function During Organ Preservation Thomas Puehler, MD, Otto Gleich,

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Impact of Normothermic Perfusion and Protein Supplementation on Human Endothelial Cell Function During Organ Preservation Thomas Puehler, MD, Otto Gleich, PhD, Simon Schopka, MD, Leopold Rupprecht, MD, Stephan Hirt, MD, Christof Schmid, MD, Karla Lehle, PhD The Annals of Thoracic Surgery Volume 89, Issue 2, Pages 512-520 (February 2010) DOI: 10.1016/j.athoracsur.2009.10.037 Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Impact of cold preservation on the mitochondrial activity (A) and the ATP content (B) in HSVEC. The HSVEC were pretreated with CMS (= control), NaCl, UW, and HTK for 2 and 6 hours (preservation). After 6 hours solutions were replaced with CMS and cells were cultivated under standard culture conditions (24 and 48 hours reperfusion). The MTS absorbance and ATP content was determined as described in the Appendix. (*p < 0.05 comparing different incubation times; # comparing different solutions.) The Annals of Thoracic Surgery 2010 89, 512-520DOI: (10.1016/j.athoracsur.2009.10.037) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Impact of warm preservation on the mitochondrial activity and cell count. The HSVEC were preserved with CMS (A), NaCl (B), HTK (C), and UW (D). After 6 hours solutions were replaced with CMS and cells were cultivated under standard culture conditions (24 and 48 hours reperfusion). The MTS absorbance (open symbols) and cell count per 96-well (filled symbols) were determined as described in the Appendix. (*p < 0.05 comparing different incubation times.) The Annals of Thoracic Surgery 2010 89, 512-520DOI: (10.1016/j.athoracsur.2009.10.037) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Impact of warm preservation on the intracellular ATP content. The HSVEC were preserved for 6 hours with CMS, NaCl, HTK, and UW and reperfused with CMS for an additional 24 and 48 hours. For experimental details see the Appendix. (*p < 0.05 comparing 6 hour preservation and 24 hour reperfusion after UW-treatment; # comparing different solutions.) The Annals of Thoracic Surgery 2010 89, 512-520DOI: (10.1016/j.athoracsur.2009.10.037) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Impact of cold (A) (B), and warm (C) (D) preservation on the expression of E-selectin (A) (C) and adhesion of PBMC (B) (D). The HSVEC were preserved with CMS, HTK, and NaCl (6 hours), reperfused with CMS for an additional 48 hours, and treated with TNF or PBS. (#p < 0.05 comparing basal and TNF-induced responses; *comparing with maximal TNF response of control cells.) The Annals of Thoracic Surgery 2010 89, 512-520DOI: (10.1016/j.athoracsur.2009.10.037) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Apoptose-induced activity of UW-treated cells. The HSVEC were preserved (6 hours) with warm (A) and cold (B) CMS (circles) and UW (diamonds), reperfused with CMS (48 hours), fixed, and DAPI stained. (Filled symbols = amount of viable cells; closed symbols = amount of apoptotic nuclei.) (C) Six hours in warm CMS; (D) 6 hours in warm UW; (E) reperfusion after warm CMS preservation; (F) reperfusion after warm UW treatment. (Magnification [40×]). The Annals of Thoracic Surgery 2010 89, 512-520DOI: (10.1016/j.athoracsur.2009.10.037) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 Mitochondrial activity (MTS absorbance) in HSVEC after normothermic (37°C) preservation (A), (D), (G) in solutions with serum proteins and reperfusion in culture medium at 37°C for 24 hours (B), (E), (H) and 48 hours (C), (F), (I). NaCl (A), (B), (C), HTK (D), (E), (F), and UW (G), (H), (I) were spiked with 4% and 10% bovine serum albumin (BSA) and human serum (HS). The ECs were stored in CMS (black bars) and spiked preservation solutions (white and patterned bars) for 6 hours; medium was exchanged and cells were incubated in CMS at 37°C (reperfusion) for 24 and 48 hours. The Annals of Thoracic Surgery 2010 89, 512-520DOI: (10.1016/j.athoracsur.2009.10.037) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions